Voltage-gated ion channels are ubiquitous proteins that orchestrate electrical signaling across excitable membranes. Key to their function is activation of the voltage sensor domain (VSD), a transmembrane four alpha-helix bundle that triggers channel opening. Modeling of currents from electrophysiology experiments yields a set of kinetic parameters for a given channel, but no direct molecular insight. Here we use molecular dynamics (MD) simulations to determine the free energy landscape of VSD activation and to, ultimately, predict the time evolution of the resulting gating currents. Our study provides the long-sought-for bridge between electrophysiology and microscopic molecular dynamics and confirms, as already suggested on the basis of experiments, that rate-limiting barriers play a critical role in activation kinetics. IntroductionDespite the complexity of the molecular processes underpinning electrical excitability of cells, the macroscopic currents measured in a typical electrophysiology experiment obey remarkably simple mathematical laws expressed in terms of few state variables. This observation, originating from the pioneering work of Hodgkin and Huxley, is replete with deep consequences: voltage gated ion channels (VGCs), the membrane proteins responsible for such currents, ought to cycle through a small number of distinct structural states. Accordingly, electrophysiology experiments are usually interpreted using the discrete state Markov (DSM) models approach, whereby a set of parameters characterizes each VGC under given conditions (1, 2). This kinetic "discreteness" has been traditionally interpreted as the consequence of few well-defined free energy basins separated by rate-limiting free energy barriers. Identifying the structure associated to each of these minima is a long-standing scientific endeavor that has benefitted from the contribution of various approaches such as mutagenesis experiments (e.g. charge reversal and disulfide locking) (3-6), X-ray crystallography (7,8) and MD simulations (9-12). The significance of these studies that relate the electrical properties of VGCs to their physico-chemical determinants (4,9,(12)(13)(14)(15)) is clear: a molecular level picture is key to understand the modulatory role of a myriad of physiological factors and to develop novel modulators.

Although the structure of TRPV1 has been experimentally determined in both the closed and open states, very little is known about its activation mechanism. In particular, the conformational changes occurring in the pore domain and resulting in ionic conduction have not been identified yet. Here, we suggest a hypothetical molecular mechanism for TRPV1 activation, which involves the rotation of a conserved asparagine in S6 from the S4-S5 linker toward the pore. This rotation is correlated with the dehydration of four peripheral cavities located between S6 and the S4-S5 linker and the hydration of the pore. In light of our hypothesis, we perform bioinformatics analyses of TRP and other evolutionary related ion channels, analyze newly available structures and re-examine previously reported water accessibility and mutagenesis experiments. Overall, we provide several independent lines of evidence that corroborate our hypothesis. Finally, we show that the proposed molecular mechanism is compatible with the currently existing idea that in TRPV1 the selectivity filter acts as a secondary gate.

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